combined treatment with low concentrations of decitabine and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 659254 11 pageshttpdxdoiorg1011552013659254

Research ArticleCombined Treatment with Low Concentrations ofDecitabine and SAHA Causes Cell Death in Leukemic Cell Linesbut Not in Normal Peripheral Blood Lymphocytes

Barbora Brodskaacute Aleš Holoubek Petra Otevlelovaacute and Katelina KuDelovaacute

Institute of Hematology and Blood Transfusion U Nemocnice 1 12820 Prague 2 Czech Republic

Correspondence should be addressed to Barbora Brodska brodskauhktcz

Received 11 April 2013 Revised 28 June 2013 Accepted 13 July 2013

Academic Editor Francesco Onida

Copyright copy 2013 Barbora Brodska et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Epigenetic therapy reverting aberrant acetylation or methylation offers the possibility to target preferentially tumor cells andto preserve normal cells Combination epigenetic therapy may further improve the effect of individual drugs We investigatedcombined action of demethylating agent decitabine and histone deacetylase inhibitor SAHA (Vorinostat) on different leukemic celllines in comparison with peripheral blood lymphocytes Large decrease of viability as well as huge p21WAF1 induction reactiveoxygen species formation and apoptotic features due to combined decitabine and SAHA action were detected in leukemic celllines irrespective of their p53 status while essentially no effect was observed in response to the combined drug action in normalperipheral blood lymphocytes of healthy donors p53-dependent apoptotic pathway was demonstrated to participate in the wtp53CML-T1 leukemic cell line response while significant influence of reactive oxygen species on viability decrease has been detectedin p53-null HL-60 cell line

1 Introduction

Contemporary cancer therapy should fulfill requirements fortargeted elimination of cancer cells simultaneously withmin-imal adverse effects Methylation and acetylation of specificsites modulate chromatin structure and intensity of geneand protein expression levels and subsequently they regulatecellular pathways involved in cell cycle control and apoptosisEpigenetic aberrations occur frequently in tumorigenesis [1ndash3] and genes silenced by abnormalmethylation or acetylationare promising targets for cancer therapy approaches [4 5]

Different DNA methyltransferases (DNMTs) ensureproper DNA methylation with different specificity forunmethylated or hemimethylated DNA DNMT1 predomi-nantlymethylates hemimethylatedCpGdinucleotides duringthe S phase maintaining the methylation pattern in the newlysynthesized strand [6] The role of DNMT3a and b is mainlyin de novo DNA methylation [7] DNMT1 expression isupregulated in certain malignant blood cells [8 9] andDNMT3A mutation is the most frequent novel genomic

variation in acute myeloid leukemias (AMLs) identified andcharacterized by parallel sequencing technologies [10] Itappears that the ldquomaintenance DNA methylationrdquo refers tothe preservation of average levels of DNA methylation atcertain regions but not to an accurate copying of site-specificDNA methylation patterns [11] DNMT1 was shown to bindp53 and to cooperate in antiapoptotic gene survivin promotermethylation in wt HCT116 cells but not in p53 null cells [12]Methyltransferase inhibitors that competitively bind to thecatalytic site of DNMT such as 5-azacytidine (Vidaza) havebeen successfully used in clinical trials to treat myelodys-plastic syndrome (MDS) [13] Deoxyribonucleotide analog5-aza-21015840-deoxycytidine (decitabine Dacogen DAC) signif-icantly reduced global methylation compared with pretreat-ment baseline in cells of AML patients [14] DAC-inducedp53 and cell cycle arrest in G2M phase have been reported inmouse embryonic fibroblasts (MEFs) with wtp53 while p53-null MEFs underwent apoptosis characterized by increaseof cell fraction in subG1 phase and caspase 3 fragmentation[15]

2 BioMed Research International

Acetylation equilibrium is maintained by balanced ratiobetween histone acetylases (HAT) and histone deacetylases(HDACs) action The activity of histones and many nonhi-stone proteins is regulated by the extent of acetylation oftheir lysine residues Dysfunction of acetylation process isoften associated with several diseases especially cancer andhistone deacetylase inhibitors (HDACi) are used to epige-netically correct aberrant HDAC activity [16 17] Changes ingene transcription direct induction of apoptosis productionof reactive oxygen species and induction of cell cycle arresthave been proposed as the mechanisms of HDACi action[18] Cell cycle arrest in G1 phase is widely documented asa consequence of HDACi-induced acetylation and transcrip-tion activation of p21WAF1 [19ndash21] The acetylation status ofp53 is extensively studied in connection with proapoptoticfunction of HDACi loss of acetylation completely abolishedp53-dependent growth arrest and apoptosis in HCT116 cells[22 23]

Methylation and acetylation mechanisms are often inter-connected and they occur ubiquitously depending on oneanother DNMT1 interacts with methyl-CpG binding pro-teins like MeCP2 which specifically recognizes fully methy-lated CpG sites or with MBD3 both forming complexeswith histone deacetylases HDAC1 and HDAC2 which inturn interact with DNMT1 [24 25] DNMT1 inhibitionwas also tested experimentally in cancer cells by alternatepathways using HDAC inhibitors that indirectly promoteubiquitin-dependent proteasomal degradation of DNMT1[26] Another mode of action of demethylating agents rep-resents the release of HDACs from gene promoters resultingin transcription activation This mechanism is responsiblefor cyclin-dependent kinase inhibitor p21WAF1 induction inAML-derived cells [27]Therefore combined action of drugsregulating methylation and acetylation is worth intensiveinvestigation

Combined treatment using two or more drugs is believedto bringmore targeted effect in cancer therapy Assessment ofconcurrent action of DNA methyltransferase inhibitor DACand histone deacetylase inhibitor suberoylanilide hydrox-amic acid (SAHA Vorinostat) showed cooperation betweenthese two drugs in suppressing colon carcinoma metasta-sis [28] or synergistic inhibition of Hey and SKOv3 cellgrowth by apoptosis and cell cycle arrest [25] Howeverlittle is known about apoptotic proteins expression dur-ing combined treatment Effect of such treatment on cellsoriginating from nonsolid tumors has not been tested yetalthough several clinical trials estimating combined effect ofDAC and SAHA administered concurrently or sequentiallyon different forms of MDS AML or ALL are currentlyin progress (httpwwwclinicaltrialsgov) In our previouswork we observed augmented effect of DAC and SAHA incombination on cell proliferation reactive oxygen speciesformation and apoptosis in leukemic cell line CML-T1 [2930] However drug concentrations used in this researchhave been shown to dramatically affect also the viabilityof peripheral blood lymphocytes (PBL) of healthy donorsTherefore action of low-drug concentrations inducing noor little adverse effect on normal cells has been studied inthis work The extent of apoptosis cell cycle distribution

p21WAF1 expression changes ROS formation and apoptoticproteins expression were investigated in leukemic cell lineswith different p53 status and in normal PBLThe mechanismof combined DAC and SAHA treatment with respect to thetumor suppressor p53 presence is discussed

2 Materials and Methods

21 Cell Culture and Chemicals Peripheral blood lympho-cytes of healthy donors were isolated from buffy coats onHistopaque 1077 (Sigma-Aldrich) as described earlier [31]Leukemia cell line CML-T1 and HL-60 (both from GermanCollection of Microorganisms and Cell Cultures Braun-schweig Germany) were cultivated in RPMI 1640 (BiochromAG Germany) supplemented with 10 FCS 37∘C and 5CO2atmosphere Decitabine (Sigma-Aldrich) and SAHA

(Cayman Chemical Company Ann Arbor MI USA) wereadded from 1mM stock solution to final concentration of1 120583M for different times up to 48 h Cytidine analogs azacyti-dine and decitabine are referred to be unstable in suspension[32] Therefore we investigated two modes of DAC actionin cell lines ldquoone-shotrdquo addition to final concentration of1 120583M for 48 h or sequential addition with fresh DAC addedafter 24 h Moreover two DAC concentrations were used in24 h sequential mode 05120583M to reach the same total drugexposure as with one-shot added 1120583M DAC or 1120583M tomaintain 1 120583M DAC concentration assuming that unstabledecitabine degraded in the first 24 h N-Acetyl-L-cysteine(NAC Sigma-Aldrich) and caspase inhibitors z-VAD-fmk(Santa Cruz) and Q-VD-OPh (RampD Systems) were added tofinal concentration of 10mM (NAC) 20120583M (z-VAD-fmk)and 10 120583M (Q-VD-OPh) respectively

22 Viability Test The viability (metabolic activity) of cellspreincubated in microplates with DAC andor SAHA for24 and 48 h was monitored using an MTT Kit I (RocheDiagnostics Corporation Indianapolis IN USA) Follow-ing the procedure described earlier [31] the absorbance ofreporter substrate was measured on an ELISA reader (MTXLab Systems Inc Vienna VA USA)

23 Flow Cytometry Distribution of cell cycle phases (mon-itored by propidium iodide PI) generation of reactive oxy-gen species (ROS observed by dichlorodihydrofluoresceindiacetate H

2DCFDA) mitochondrial membrane potential

(MMP measured by MitoTracker Red MTR) condensa-tion of chromatin (DNA stained by Hoechst 33342) andthe extent of apoptosis (defined by Annexin V-FITC posi-tivePI negative population of treated cells) were investigatedby flow cytometry All fluorescent probes were purchasedfrom Life Technologies Corporation All samples (50 000eventssample)were analyzed on anLSRFortessa cell analyzer(BD Biosciences)

231 Cell Cycle Cells treated by DAC andor SAHA for 24or 48 h were harvested washed twice with 2mL of PBSresuspended in 500120583L of PBS added into 45mL of ice-cold70 EtOH and stored in minus20∘C For the analysis suspension

BioMed Research International 3

was washed with PBS resuspended in PI-staining solution(01 (vv) Triton X-100 100120583gmL RNaseA 50 120583gmL PI)and kept for 30min in the dark

232 ROS Production and Mitochondrial Membrane Poten-tial Treated cells were harvested washed with PBS and sus-pended in 1mL PBS H

2DCFDA MTR and Hoechst 33342

were added to final concentration of 10 120583M (H2DCFDA)

40 nM (MTR) and 2 120583M (Hoechst 33342) for 30min in 5CO2at 37∘C

233 Apoptosis Cells were harvested washed with PBS andresuspended in 100 120583L of Annexin Binding Buffer (ABB10mM Hepes pH 74 140mM NaCl and 25mM CaCl

2)

containing 5 120583L of Annexin V-FITC solution After 15min inthe dark (RT) 400 120583L of ABB was added and suspension wasincubated for another 5min with 2120583L of 250120583gmL PI stocksolution

24 RNA Isolation and qRT-PCR RNA of total 5 times 106cells was isolated with RNeasy kit (Qiagen) according tomanufacturers instructions and deposited in minus80∘C untiluse mRNA quality and concentration were assessed on ND-1000 Nanodrop system and qRT-PCR was performed onCFX96 real-time system (Bio-Rad) using SensiFAST SYBRNo-ROX One-Step Kit (Bioline) Relative mRNA quantityand standard deviations were calculated with CFX ManagerSoftware 120573-Actin was used as reference gene Primers for RT-PCR were designed with Primer-BLAST software (NCBI)

25 Immunoblotting For immunoblotting two ways of sam-ple collection were used The first method efficiently pre-vented unstable protein degradation and involved cell lysisdirectly to the Laemmli sample buffer This procedure pre-cluded quantification of protein concentration by Bradfordreagent and it was used for samples from PBL whose stabilityin usual lysis buffer was poor and its protein concentrationwas supposed to be stable and for experiments with caspaseinhibitors For protein expression analysis from cell linesstandard lytic procedure was used cells were lysed in LyticBuffer (LB 50mM Tris HCl pH 75 1 NP-40 5mM EDTA150mM NaCl 1mM DTT 1mM PMSF 4120583L1mL proteaseinhibitor cocktail and 5120583L1mL phosphatase inhibitor cock-tail 2 both cocktails were from Sigma-Aldrich) for 30minat 4∘C and then centrifuged at 10000 g30min4∘C Afterprotein concentration assay (Bradford) supernatants weremixed with 2xLaemmli sample buffer and boiled for 5minFive micrograms of total protein were then subjected toSDS-PAGE and transferred into nitrocellulose membrane(Hybond PVDF Amersham) Rabbit polyclonal primaryantibodies against Bcl-2 Bcl-xL BidMcl-1 p21 PARP Pumaand survivin as well as mouse monoclonal anti-p53 andanti-caspase-3 (procaspase and active form) were from SantaCruz Biotechnology Mouse monoclonal anti-Bax originatedfrom BioLegend and mouse monoclonal antibodies againstcaspases-7 and -8 (procaspase and active form) IAP Smacand XIAP were from BD Biosciences Mouse monoclonalanti-120573-Actin (Sigma-Aldrich) was used to detect 120573-Actin

expression as control of equal loading All primary antibod-ies except anti-PARP (1 1000) and anti-120573-Actin (1 5000)were used at dilution 1 500 Anti-rabbit and anti-mouseHRP-conjugated secondary antibodies were purchased fromThermo Scientific and used in concentrations 1 50000ndash1 100000 ECL Plus Western Blotting Detection System(Amersham) was used for chemiluminescence visualizationand evaluation by G-box iChemi XT4 digital imaging device(Syngene Europe Cambridge) Relative protein expressionwas evaluated by GeneTools Software and statistical analysiswas performed in GraphPad software

26 Fluorescence Microscopy Cells in suspension wereseeded on coverslip in humidified chamber for 15min andthen fixed with 4 paraformaldehyde (PFA) overnightAfter 10min of permeabilization by 05 Triton X-100the cells were incubated for 1 h with mouse monoclonalanti-Bax (BioLegend) and rabbit polyclonal anti-COX (CellSignaling Technology) primary antibodies (1 100) and foranother 1 h with the mixture of secondary antibodies(Alexa Fluor488-conjugated anti-rabbit and Alexa Fluor647-conjugated anti-mouse both from Life Technologies 1 200)with Hoechst33342 (1120583M Life Technologies) After extensivewashing by PBS-Tween stained cells were mounted intoProLong mounting suspension and observed under confocallaser scanning microscope FluoView FV1000 (Olympus Cor-poration) Fluorescence images were processed by FluoViewsoftware FV10-ASW 31

27 Statistical Analysis In diagrams arithmetic means ofat least four times repeated experiments were plotted withSEM error bars Significance levels (119875 values of 119905-tests) weredetermined using InStat Software (GraphPad Software) A119875 value of 005 or lower was considered to be a statisticallysignificant difference between groups compared

3 Results

31 Cell Viability The effect of 1 120583M DAC andor 1120583MSAHA on cell viability has been investigated during 48-hourtreatment (Figure 1) In the 24 h time frame DAC alone aswell as SAHA caused slight viability decrease in CML-T1 cellline Similar decrease after DAC treatment has been observedin p53-null HL-60 cells whereas no effect of SAHA wasdetected inHL-60 cell lineWhile the viability of lymphocyteswas maintained at the control level even after 48 h of DAC +SAHA combination treatment both cell lines wtp53 CML-T1 and p53-null HL-60 showed significant viability decrease(119875 lt 0001)

32 Apoptotic Markers Annexin V+PIminus cell fraction PARPfragmentation executive caspases 37 activation mitochon-drial membrane depolarization and fraction of cells in subG1phase were analyzed to determine the extent of apoptosisinduced by DAC andor SAHA treatment (Figure 2)

In all these apoptotic parameters moderate changescaused by individual agents in CML-T1 or no apoptotic


0 10 20 30 40 50 600

20

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Time (h)

Viab

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()

CML-T1

(a)

0 10 20 30 40 50 600

20

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Time (h)

Viab

ility

()

HL-60

(b)

0 10 20 30 40 50 600

20

40

60

80

100

120

Time (h)

Viab

ility

()

PBL

(c)

Figure 1 Effect of DAC andor SAHA on cell viability during 48-hour treatment Cells of leukemic cell lines CML-T1 (a circles) and HL-60(b squares) or PBL (c triangles) were treated by 1120583M DAC (full line) 1120583M SAHA (dash line) or 1 120583M DAC + 1 120583M SAHA (dotted line) inthe 48 h time frame and then their metabolic activity was measured by MTT test Data are the average of at least five experiments and plusmnSEMare plotted as error bars

effect of SAHA on HL-60 cells in contrast to large apop-totic effect after 48 h of concurrent drugs action have beendetected in both cell lines Annexin V positivity was notinfluenced by 20120583M caspase inhibitor z-VAD-fmk but itwas efficiently attenuated by 10 120583M Q-VD-OPh Increaseof Annexin V+PI+ cell population induced by caspaseinhibitors was observed in HL-60 cell line concurrentlywith Annexin V+PIminus fraction lowering (data not shown)This effect indicates damaging action of DAC and SAHAwhich is not possible to reverse by caspase inhibition in HL-60 cell line In CML-T1 cells both used caspase inhibitorswere able to effectively reduce caspase activation (Figure 3)causing 40 (z-VAD-fmk) and 60 (Q-VD-OPh) attenua-tion of active caspase-7 fragment respectively and 85 (z-VAD-fmk) respectively complete (Q-VD-OPh) inhibition ofactive caspase-3 fragment On the caspase-3 immunoblotseveral intermediate products were visible in the presenceof inhibitors We did not detect any Annexin positivitycaspases activation or subG1 peak in PBL (data not shown)Slight membrane depolarization and PARP fragmentation

were observed after SAHA treatment of PBL but these effectswere not augmented by DAC addition

33 Cell Cycle Arrest and p21 Induction Distribution of cellcycle phases was monitored by PI staining of ethanol-fixedcells during 48 h DAC andor SAHA treatment (Figure 4(a))Simultaneously expression of cyclin-dependent kinaseinhibitor p21WAF1 which is tightly connected with cell cycleprogression from G1 phase was examined (Figure 4(b))Individual phases proportion was difficult to determine inCML-T1 cells due to their nearly tetraploid karyotype withdiploid sideline Nevertheless p21WAF1 expression wassubstantially enhanced in all cases that is after DAC andorSAHA treatment of CML-T1 cells Decitabine increasedG2-phase cell fraction while SAHA caused an arrest in G1phase in HL-60 cell line The effect of combined treatment ismainly in large subG1 phase increase but high proportion ofnonapoptotic cells was found in G1 phase after DAC + SAHAtreatment Huge p21 induction was observed in both SAHA-and DAC + SAHA-treated samples but increased p21WAF1


CML-T1 HL-600

Frac

tion

of ce

lls (

)

5

10

15

20

25 lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(a)

CML-T1 HL-60

Frac

tion

of ce

lls (

)

0102030405060

lowast

lowastlowastlowast

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(b)

CML-T1 HL-60

Frac

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of ce

lls (

)

0

50

100 lowastlowast


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(c)

Caspase-3

Caspase-7

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Procaspase

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Active caspase

CML-T1 HL-60

SAHA

DACminus

minus minus

+ minus + minus + minus

minusminus

+

++++

(d)

PARP

PBL PARP f116

f116

f88

f88

SAHADAC

CML-T1 HL-60

SAHADACminus

minus minus


minusminus

+

++++

minus

minus minus

+ minus +

++

(e)

Figure 2 Apoptotic features induced by DAC andor SAHA treatment appropriate fraction of control cells (empty) or cells treated for 48 hby 1 120583MDAC (vertically shaded) 1120583M SAHA (horizontally shaded) or DAC + SAHA combination (filled) measured by flow cytometry (a)AnnexinV-FITCPI staining of nonfixed cells AnnexinV+PIminus fraction effect of 10 120583Mz-VAD-fmk (dotted) or 20120583MQ-VD-Oph (checked)(b) PI staining of ethanol-fixed cells fraction of cells in subG1 phase (c) MitoTracker Red fluorescence fraction of cells having polarizedmitochondrial membrane Error bars represent plusmnSEM of at least 3 measurements Statistical significance degree of difference between treatedsamples and the corresponding control 119875 lt 005 (lowast) 119875 lt 001 (lowastlowast) and 119875 lt 0001 (lowastlowastlowast) (d) Immunoblots showing caspases-3 and -7activation after 48 h DAC andor SAHA treatment (e) Fragmentation of PARP in CML-T1 and HL-60 cell lines or PBL treated by DACandor SAHA for 48 h Images are representatives of at least three experiments

Caspase-3

Caspase-7

Procaspase

Procaspase

Active caspase

Active caspase

SAHADAC

Q-VD-OPhz-VAD-fmk

minus

minus minus

minusminus minus minus minus

minus minus minus minus minus

minus+ + + +

+ +

+

+

+ +

Figure 3 Inhibition of executive caspases cleavage by caspaseinhibitors z-VAD-fmk (20120583M) or Q-VD-OPh (10120583M) in CML-T1cells treated by 1120583MDAC + 1 120583M SAHA for 48 h

expression was detected also in DAC-treated HL-60 cells Nochanges were observed in lymphocytes which were almostcomplete in G0G1 phase the proportion of subG1 cellswas up to 2 in PBL (data not shown) in all samples andexpression of p21WAF1 was maintained at the same level inall PBL tested samples

34 ROS Production Production of reactive oxygenspecies was monitored by H

2DCFDA fluorescence intensity

(Figure 5) Surprisingly only slight ROS production wasdetected in any cells treated by 1 120583MSAHA alone Decitabinealone or in combination caused H

2DCFDA fluorescence

distribution peak broadening but only moderate meanfluorescence value shift in CML-T1 cells Contrary to this cellline in HL-60 cells DAC alone or in combination induced apopulation with high H

2DCFDA fluorescence intensity and

multicolor staining showed that this population is identicalwith the population of cells with depolarized mitochondrialmembrane and condensed chromatin (more intensiveHoechst staining) Addition of 10mM N-acetyl-L-cysteine(NAC) lowered the DAC-induced ROS production in bothcell lines but in CML-T1 NAC simultaneously inducedmitochondrial membrane depolarization in comparisonwith control cells without NAC Again there were noconsiderable changes in ROS production in lymphocytes

35 Mode of DAC Addition In CML no substantial differ-ence between the samples with 05120583M and 1120583M DAC wasobserved and the effect was comparable also for sequentialDAC addition versus one-shot mode Effect of sequentialDAC addition into HL60 cell suspension either with orwithout SAHA was strongly influenced by DAC concen-tration 05120583M repeated DAC caused significantly lowereffect (119875 lt 005) in all parameters (cell viability decreasemitochondrial membrane depolarization fraction of cellsin subG1 phase PARP fragmentation executive caspasesactivation ROS generation and p21 expression) than 1120583M


SubG1 G2G1

0

20

40

60

80

100

C D S

S

DS C D S DS

Cel

l cyc

le p

hase

frac

tion

()

CML-T1 HL-60

(a)

p21

PBL p21

SAHADAC

CML-T1 HL-60

SAHA

DACminus

minus

+

+

minus

minus

+

minus + minus +

+

minus +minus +

minus +minus +

minus + minus +

120573-Actin

120573-Actin

(b)

Figure 4 (a) Distribution of cell cycle phases monitored by PI staining of ethanol-fixed cells untreated (C) or treated by 1120583M DAC (D)1 120583M SAHA (S) or DAC + SAHA combination (DS) Data are averaged from at least three experiments (b) Expression of cyclin-dependentkinase inhibitor p21WAF1 after 48 h of 1 120583MDAC andor 1 120583M SAHA treatment of leukemic cell lines (CML-T1 HL-60) or PBL Images arerepresentatives of at least five experiments

CML-T1 HL-600

1

2

3

4

Mea

n flu

ores

cenc

e int

ensit

y (a

u)

lowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

Figure 5 Production of reactive oxygen species Control cells(empty) and cells treated for 48 h by 1120583MDAC (vertically shaded)1 120583M SAHA (horizontally shaded) or DAC + SAHA combination(filled) were stained by H

2DCFDA and the mean value of flow-

cytometer-detected fluorescence intensity was analyzed 10mMNACwas used forDAC+ SAHA-induced ROS generation reduction(checked) Error bars represent plusmnSEM of four measurementsStatistical significance degree of difference between treated samplesand the corresponding control 119875 lt 005 (lowast) 119875 lt 001 (lowastlowast) and119875 lt 0001 (lowastlowastlowast)

DAC On the other hand 1 120583MDAC induced nearly identicaleffect independently of the mode of addition in HL60 cellsFraction of cells in subG1 phase and executive caspasesactivation are presented in Figure 6 as representative resultsof these phenomena

36 mRNA Expression The transcription levels of cyclin-dependent kinase inhibitor p21WAF1 tumor suppressor gene

TP53 and several apoptosis-related genes were investigatedby qRT-PCR (Figure 7)Massive p21WAF1 gene induction hasbeen observed in both individual agents and combination-treated cell lines Expression of TP53 was not influenced byDAC it was lowered by SAHA or combination in CML-T1and of course not detectable in p53-null HL-60 cells Genesfor proapoptotic proteins Bax and Puma were induced byDAC in CML-T1 but even decreased in HL-60 where Bcl-2 gene was also attenuated after DAC + SAHA treatmentSurprisingly Mcl-1 induction was observed namely in HL-60 cells indicating activation of some antiapoptotic mech-anism No statistically significant changes were detected intranscription levels of the antiapoptotic protein Bcl-xL

37 Protein Expression Expression of tumor suppressor p53and proteins related to apoptosis was studied by immunoblot(Figure 8) p53 expression was induced by DAC in CML-T1cells independently on concurrent SAHA presence Togetherwith p53 induction increase of p53-upregulated mediator ofapoptosis Puma expression was observed No p53 expressionchange has been documented in lymphocytes and of coursein p53-null HL-60 cell line where Puma level diminutionwas simultaneously detected Contrarily slight decrease ofantiapoptotic protein Bcl-2 level was detected after combinedtreatment of HL-60 but not in CML-T1 Induction of Mcl-1observed on transcription level was detected also in proteinexpression Onlyminimal increase of protein expression aftertreatment was detected for proapoptotic Bax We did notdetect any change in expression of several other apoptosis-related proteins Bcl-xL BIM IAP XIAP Smac and survivin

38 Fluorescence Microscopy Activation of proapoptotic Baxduring its translocation from cytoplasm into mitochondrial


CML-T1 HL-60

0

5

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actio

n of

cells

()

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of ce

lls (

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lowast

C D

DD

1

DD

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SAH

A

SAH

A+

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2 C D

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DD

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A

SAH

A+

DSA

HA+

DD

1SA

HA+

DD

2

(a)

CML-T1 HL-60

Procaspase

Procaspase

SAHA

Caspase-7

Caspase-3

1120583M DAC05120583M DAC

1120583M DAC after 24h


32kDa intermediate

17kDa fragment

minus

minus minus minus minus

minus

minus

minus minus

minus minus minus minusminus

minus

minus

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+

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+

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(b)

Figure 6 Apoptotic effects of one shot versus sequential DAC treatment (a) Control cells (C) were treated by 1 120583M DAC for 48 h (D) orsequentially treated by two doses of 05120583M DAC (DD1) or 1 120583M DAC (DD2) every 24 h in the presence or absence of 1120583M SAHA Treatedcells were fixed in EtOH and stained by PI and the fraction of cells in subG1 phase of cell cycle was analyzed Error bars represent plusmnSEMof four measurements Statistical significance degree of difference 119875 lt 005 (lowast) (b) Immunoblots showing executive caspases activation insamples are described in (a)

CML-T1

p21 p53 Puma BAX Bcl2 Mcl10

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ive e

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(b)

Figure 7 Transcription levels of cyclin-dependent kinase inhibitor p21WAF1 tumor suppressor gene TP53 and several apoptosis-relatedgenes in leukemic cell lines treated by 1120583M DAC andor 1 120583M SAHA monitored by qRT-PCR Error bars represent plusmnSEM of threemeasurements Statistical significance degree of difference between treated samples and the corresponding control 119875 lt 005 (lowast) 119875 lt 001(lowastlowast) and 119875 lt 0001 (lowastlowastlowast)


p53

Puma

CML-T1 HL-60

SAHADAC

Bcl2

Mcl-1

BAX

minus minus+ + minus + minus

minusminus

+

++minusminus ++

120573-Actin

Figure 8 Expression of tumor suppressor p53 and proteins relatedto apoptosis in control cells and after 48 h DAC SAHA or DAC +SAHA treatment of leukemic cell lines monitored by immunoblot-ting Images are representatives of at least four experiments withsimilar results

membrane is widely described as one step of the intrinsicapoptotic pathway EnhancedBax gene expression inCML-T1cells was not reflected by substantial increase of Bax proteinlevel but fluorescence microscopy revealed redistributionof Bax from whole cellular volume into mitochondria inapoptotic cells (Figure 9) On the other hand Bax localizationin HL-60 was nearly mitochondrial even in intact cells andconcentration of fluorescence signal into mitochondria inapoptotic cells after DAC andor SAHA treatment was notaccompanied by significant fluorescence intensity change(data not shown)

4 Discussion

The effectiveness of chemotherapy drugs may be improvedby administering them in combination (combination chemo-therapy) Combination drug therapies can target multiplepathologic processes and lower drug doses allow for min-imizing the adverse side effects Moreover de novo andacquired resistance arises with molecularly targeted drugsand cytotoxic chemotherapy limiting their utility and ratio-nal combinatorial targeted therapy can help to overcome thisproblem [33]

Many combination therapies are used to treat metastaticbreast cancer [34] Several inhibitor combinations effectivefor melanomas with activating RAS or BRAFmutations wererecently discovered [35] Combination therapy of 120572-galacto-sylceramide and 5-fluorouracil showed antitumor effect inmice and suggests a new therapy mode against metastaticliver cancer [36] More pronounced effect of rapamycinand docetaxel combination has been manifested againstnonsmall-cell lung cancer [37] However several combi-nation therapies revealed excessive adverse effect and badtolerability [38] or were not improving effect of single drugs[39] In this paper combination of drugs affecting two fun-damental epigenetic processes methylation and acetylationhas been investigated on cell lines originating from leukemia

patients The effect on wtp53 CML-T1 line and p53-null HL-60 line was compared with the effect on PBL of healthydonors The viability assay manifested no effect of individualDACor SAHA treatment on PBL which remained unaffectedeven when combined treatment is applied By contrastmarked viability drop after 48 h of combined DAC + SAHAtreatment has been detected in both cell lines tested irrespec-tive of their p53 status surpassing the individual drug effectsSeveral apoptotic features were further analyzed to determinethe extent of apoptosis induced by individual drugs and theircombination on the cell lines with different p53 status Whileannexin V positivity PARP fragmentation and caspases-3and -7 activation correlated with viability drop observed inCML-T1 cells all these changes had only moderate extent inHL-60 cells Caspase inhibitors effectively blocked caspases-37 cleavage but even increasing apoptotic cells fractionin Annexin V-FITC staining was observed using 20120583Mz-VAD-fmk inhibitor It is likely that z-VAD-fmk-inducedinhibition of executive caspases fragmentation is not reflectedby entire recovery of cellular processes and cells are directedto cell death despite blocked caspases Indeed unspecificdamaging effect of this inhibitor was recently observed inrenal injury [40] On the other hand comparable extentof mitochondrial membrane depolarization was detected inboth cell lines tested Most of the parameters tested remainedunchanged after DAC andor SAHA treatment of PBL Theonly effect of SAHA alone or in combination with DACon PBL was slight mitochondrial membrane depolarizationand PARP fragmentation which was not accompanied byviability drop or executive caspases activation As epigeneticaction of DAC targets S-phase fraction of cells prevalentappearance of lymphocytes in G0G1 phase of cell cycle is thepossible reason of high lymphocyte resistance to low DACconcentration exposure

Cell cycle analysis brought information about significantproportion of cells namely of HL-60 line in subG1 phasewhen treated by DAC and SAHA While interpretation ofcell cycle distribution of CML-T1 cells was complicated bypartial tetraploidy of CML-T1 cell line cell cycle arrest inG1 after SAHA treatment and G2 phase arrest after DACactionwas evident inHL-60 lineHowever both of these indi-vidual drug effects were overlaid by marked increase of cellsin subG1 after combined drug action Cell cycle arrest isusually in tight connection with cyclin-dependent kinase(cdk) inhibitors therefore we tested the gene and proteinlevels of p21WAF1 which are known to inhibit several cdksmainly cdk2 In CML-T1 cells both gene and protein levelswere significantly enhanced after individual drug action andalso after combination treatment Large p21WAF1 expressionincrease was detected also in HL-60 treated by SAHA orby DAC + SAHA while only moderate enhancement wasobserved in DAC-treated HL-60 This is in agreement withfindings of other authors describing direct effect ofHDACi onp21WAF1 promoter acetylation and its expression induction[41 42] and p53-induced p21WAF1 activation of wtp53 cellsafter DAC treatment [43] Induction of p21WAF1 by DACeven in p53-null HL-60 cells suggests another mechanismof p21WAF1 expression stimulation One of such possiblemechanisms is reactive oxygen species (ROS) formation


Anti-BAX

Intact

Anti-COX Merge

DAC +SAHA

Figure 9 Intact cells CML-T1 and cells treated by DAC + SAHA combination for 48 h stained with anti-Bax (red Alexa Fluor647) andmitochondrial anti-COX (green Alexa Fluor488) antibodies Nuclei are visualised by Hoechst 33342 Scale bar represents 10 120583m

induced by DAC treatment occurring especially in p53-nullcells [44 45] Recently direct relation between DAC-inducedDNMT1 depletion and p21WAF1 induction was observed indrug-resistant breast cancer cells (MCF-7ADR) [46]

Both DAC and DAC + SAHA treatments induced ROSgeneration in both cell lines tested but this effect wasmore pronounced in HL-60 cell line Moreover N-acetyl-L-cysteine (NAC) protected p53-null HL-60 cells more effec-tively than wtp53 cell line CML-T1 The main mechanismof DAC action on HL-60 cells is therefore probably ROSproduction causing enhanced p21 expression and moder-ate level of apoptosis Relatively low extent of apoptoticchanges in comparison with high-treatment cytotoxicity andpersistence of PI-positive cell fraction in the presence ofcaspase inhibitors indicate that another type of cell deathis induced in parallel with the apoptosis in HL-60 cellsROS formation p21WAF1 gene and protein induction cellcycle arrest and also DAC-induced increase of cell volumeimply possible role of senescence [47ndash49] the mechanism ofDAC action which has been observed in prostate cancer cells[50] or in malignant pleural mesothelioma [51] Inductionof senescence has been detected also in SAHA-treated coloncancer cells with nonfunctioning p21WAF1 or p53 genes [52]

Although DAC is reported to have short half-life insuspension we did not detect any substantial differencebetween the samples with 1 120583MDAC that is fresh 1 120583MDACadded into treated cell suspension after 24 h of action had nosignificant effect Contrarily sequential 05120583MDAC inducedsignificantly lower effect than the 1 120583M DAC in HL60

regardless of SAHA presence No difference was detected inCML which indicates that although 1 120583MDAC induces com-parable effect in both of tested cell lines CML but not HL60line is equally sensitive even to lower drug concentrationTheeffects on apoptotic parameters are probably initiated in thefirst 24 h of DAC action before its depletion and therefore noeffect of additional drug dose has been observed

Analysis of apoptotic gene and protein expressionchanges revealed p53-dependent apoptotic way of cell deathin CML-T1 cells Attenuated degradation by proteasomeshould be the mechanism of DAC-induced boost of p53protein expression because the increase in protein level is notpreceded by TP53 gene induction Both the gene and proteinexpressions of p53-inducible protein Puma were enhanced inconcert with p53 stabilization Althoughwe detected increaseof Bax mRNA in CML-T1 only minimal change has beenobserved on active Bax protein level However fluorescencemicroscopy revealed high accumulation of active Bax inmitochondria of apoptotic cells Bax participation on apop-tosis is mediated by its relocalization into the mitochondrialmembrane [53] Our results show that this relocalization isnot necessarily accompanied by large change of overall Baxexpression

5 Conclusion

Combined treatment with epigenetic drugs was tested incell lines originating from nonsolid tumors (leukemia) withrespect to their p53 status Participation of proteins related


to apoptosis was investigated during individual as well ascombined drug treatment and multiple apoptotic featureswere monitored In summary combination of decitabineand SAHA markedly decreased the viability of leukemia celllines While in wtp53 cell line apoptosis was induced also byindividual agents alone and their effect in combination wasadditive p53-null cells seemed to be sensitized by SAHAwhich in itself was not toxic for HL-60 for enhanced reactionin response to DAC presence Tumor suppressor p53 and itsdownstream targets Puma and Bax were identified to play themain role in apoptosis in wtp53-possessing CML-T1 cell linewhile ROS formation was found to affect cell death in p53-null HL-60 cell lineThe absence of negative effect on normalperipheral blood lymphocytes nominates this combination ofanticancer drugs to the group of possible therapeutic toolsthat are profitable for further investigation

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by the Project for ConceptualDevelopment of Research Organization no 00023736 fromthe Ministry of Health Czech Republic and by the GrantERDF OPPK CZ216310024001

References

[1] V E Sollars ldquoEpigenetic modification as an enabling mecha-nism for leukemic transformationrdquo Frontiers in Bioscience vol10 no 2 pp 1635ndash1646 2005

[2] T A Chan and S B Baylin ldquoEpigenetic biomarkersrdquo CurrentTopics in Microbiology and Immunology vol 355 pp 189ndash2162012

[3] M Hatziapostolou and D Iliopoulos ldquoEpigenetic aberrationsduring oncogenesisrdquo Cellular and Molecular Life Sciences vol68 no 10 pp 1681ndash1702 2011

[4] A Ganesan L Nolan S J Crabb and G Packham ldquoEpigenetictherapy histone acetylation DNAmethylation and anti-cancerdrug discoveryrdquo Current Cancer Drug Targets vol 9 no 8 pp963ndash981 2009

[5] S Sharma T K Kelly and P A Jones ldquoEpigenetics in cancerrdquoCarcinogenesis vol 31 no 1 Article ID bgp220 pp 27ndash36 2009

[6] H P Easwaran L Schermelleh H Leonhardt and M C Car-doso ldquoReplication-independent chromatin loading of Dnmt1during G2 and M phasesrdquoThe EMBO Reports vol 5 no 12 pp1181ndash1186 2004

[7] M Okano D W Bell D A Haber and E Li ldquoDNA methyl-transferases Dnmt3a and Dnmt3b are essential for de novomethylation and mammalian developmentrdquo Cell vol 99 no 3pp 247ndash257 1999

[8] F Langer J Dingemann H Kreipe and U Lehmann ldquoUp-regulation of DNA methyltransferases DNMT1 3A and 3B inmyelodysplastic syndromerdquo Leukemia Research vol 29 no 3pp 325ndash329 2005

[9] S Mizuno T Chijiwa T Okamura et al ldquoExpression of DNAmethyltransferases DNMT1 3A and 3B in normal hema-topoiesis and in acute and chronic myelogenous leukemiardquoBlood vol 97 no 5 pp 1172ndash1179 2001

[10] L Riva L Luzi and P G Pelicci ldquoGenomics of acute myeloidleukemia the next generationrdquo Frontiers in Oncology vol 2article 40 2012

[11] R Z Jurkowska T P Jurkowski and A Jeltsch ldquoStructureand function of mammalian DNA methyltransferasesrdquo Chem-BioChem vol 12 no 2 pp 206ndash222 2011

[12] P Esteve H G Chin and S Pradhan ldquoHuman maintenanceDNA (cytosine-5)-methyltransferase and p53 modulate expres-sion of p53-repressed promotersrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no4 pp 1000ndash1005 2005

[13] L R Silverman E P Demakos B L Peterson et al ldquoRan-domized controlled trial of azacitidine in patients with themyelodysplastic syndrome a study of the cancer and leukemiagroup Brdquo Journal of Clinical Oncology vol 20 no 10 pp 2429ndash2440 2002

[14] P Yan D Frankhouser M Murphy et al ldquoGenome-widemethylation profiling in decitabine-treated patients with acutemyeloid leukemiardquo Blood vol 120 pp 2466ndash2474 2012

[15] M Nieto E Samper M F Fraga G Gonzalez De Buitrago MEsteller and M Serrano ldquoThe absence of p53 is critical for theinduction of apoptosis by 5-aza-21015840-deoxycytidinerdquo Oncogenevol 23 no 3 pp 735ndash743 2004

[16] S B Baylin and J E Ohm ldquoEpigenetic gene silencing incancermdasha mechanism for early oncogenic pathway addictionrdquoNature Reviews Cancer vol 6 no 2 pp 107ndash116 2006

[17] P P Pandolfi ldquoHistone deacetylases and transcriptional therapywith their inhibitorsrdquoCancer Chemotherapy and Pharmacologyvol 48 supplement 1 pp S17ndashS19 2001

[18] W S Xu R B Parmigiani andPAMarks ldquoHistone deacetylaseinhibitors molecular mechanisms of actionrdquoOncogene vol 26no 37 pp 5541ndash5552 2007

[19] L Bellucci M Dalvai S Kocanova F Moutahir and KBystricky ldquoActivation of p21 by HDAC inhibitors requiresacetylation of H2AZrdquo PLoS One vol 8 Article ID e54102 2013

[20] Q ZhouC LDalgard CWynder andM LDoughty ldquoHistonedeacetylase inhibitors SAHA and sodium butyrate block G1-to-S cell cycle progression in neurosphere formation by adultsubventricular cellsrdquo BMCNeuroscience vol 12 article 50 2011

[21] H Wang W Zhou Z Zheng et al ldquoThe HDAC inhibitordepsipeptide transactivates the p53p21 pathway by inducingDNA damagerdquo DNA Repair vol 11 no 2 pp 146ndash156 2012

[22] Y Tang W Zhao Y Chen Y Zhao and W Gu ldquoAcetylation isindispensable for p53 activationrdquo Cell vol 133 no 4 pp 612ndash626 2008

[23] H Yamaguchi N T Woods L G Piluso et al ldquop53 acetylationis crucial for its transcription-independent proapoptotic func-tionsrdquo The Journal of Biological Chemistry vol 284 no 17 pp11171ndash11183 2009

[24] W Qin H Leonhardt and G Pichler ldquoRegulation of DNAmethyltransferase 1 by interactions andmodificationsrdquoNucleus(Austin Tex) vol 2 no 5 pp 392ndash402 2011

[25] M Chen W S-L Liao Z Lu et al ldquoDecitabine and suberoy-lanilide hydroxamic acid (SAHA) inhibit growth of ovariancancer cell lines and xenografts while inducing expression ofimprinted tumor suppressor genes apoptosis G2M arrest andautophagyrdquo Cancer vol 117 no 19 pp 4424ndash4438 2011

[26] Q Zhou A T Agoston P Atadja W G Nelson and NE Davidson ldquoInhibition of histone deacetylases promotesubiquitin-dependent proteasomal degradation of DNAmethyl-transferase 1 in human breast cancer cellsrdquo Molecular CancerResearch vol 6 no 5 pp 873ndash883 2008


[27] S A Scott W Dong R Ichinohasama et al ldquo5-Aza-21015840-deoxy-cytidine (decitabine) can relieve p21WAF1 repression in humanacute myeloid leukemia by a mechanism involving release ofhistone deacetylase 1 (HDAC1) without requiring p21WAF1promoter demethylationrdquo Leukemia Research vol 30 no 1 pp69ndash76 2006

[28] D Yang C M Torres K Bardhan M Zimmerman T LMcGaha and K Liu ldquoDecitabine and vorinostat cooperate tosensitize colon carcinoma cells to fas ligand-induced apoptosisin vitro and tumor suppression in vivordquo Journal of Immunologyvol 188 no 9 pp 4441ndash4449 2012

[29] B Brodska and A Holoubek ldquoGeneration of reactive oxygenspecies during apoptosis induced by DNA-damaging agentsandor histone deacetylase inhibitorsrdquo Oxidative Medicine andCellular Longevity vol 2011 Article ID 253529 7 pages 2011

[30] B Brodska P Otevrelova and A Holoubek ldquoDecitabine-induced apoptosis is derived by Puma and Noxa induction inchronic myeloid leukemia cell line as well as in PBL and ispotentiated by SAHArdquoMolecular andCellular Biochemistry vol350 no 1-2 pp 71ndash80 2011

[31] I Kalousek B Brodska P Otevrelova and P Roselova ldquoActino-mycin D upregulates proapoptotic protein Puma and downreg-ulates Bcl-2 mRNA in normal peripheral blood lymphocytesrdquoAnti-Cancer Drugs vol 18 no 7 pp 763ndash772 2007

[32] C Stresemann and F Lyko ldquoModes of action of the DNAmeth-yltransferase inhibitors azacytidine and decitabinerdquo Interna-tional Journal of Cancer vol 123 no 1 pp 8ndash13 2008

[33] B Al-Lazikani U Banerji and P Workman ldquoCombinatorialdrug therapy for cancer in the post-genomic erardquo NatureBiotechnology vol 30 pp 679ndash692 2012

[34] J H Lee R M Pidaparti G M Atkinson and R S MoorthyldquoDesign of an implantable device for ocular drug deliveryrdquoJournal of Drug Delivery vol 2012 Article ID 527516 8 pages2012

[35] M A Held C G Langdon J T Platt et al ldquoGenotype-sel-ective combination therapies for melanoma identified by high-throughput drug screeningrdquoCancer Discovery vol 3 pp 52ndash672013

[36] H Aketa T Tatsumi K Kohga et al ldquoThe combinationtherapy of alpha-galactosylceramide and 5-fluorouracil showedantitumor effect synergistically against liver tumor in micerdquoInternational Journal of Cancer 2013

[37] L Xu Y Qin J Huang et al ldquoEffect of rapamycin-inducedtumor vessel thrombosis combined with docetaxel in non-small-cell lung cancerrdquo Anti-Cancer Drugs vol 24 no 4 pp406ndash414 2013

[38] H Gerullis C Eimer T H Ecke E Georgas C Arndt andT Otto ldquoCombined treatment with pazopanib and vinfluninein patients with advanced urothelial carcinoma refractory afterfirst-line therapyrdquo Anti-Cancer Drugs vol 24 no 4 pp 422ndash425 2013

[39] M S Lee H J Mamon T S Hong et al ldquoPreoperative cetux-imab irinotecan cisplatin and radiation therapy for patientswith locally advanced esophageal Cancerrdquo Oncologist vol 18no 3 pp 281ndash287 2013

[40] G P Kaushal and SV Shah ldquoNon-apoptotic effects of antiapop-totic agent zVAD-fmk in renal injuryrdquoKidney International vol83 no 3 p 531 2013

[41] C-Y Gui L Ngo W S Xu V M Richon and P A MarksldquoHistone deacetylase (HDAC) inhibitor activation of p21WAF1involves changes in promoter-associated proteins including

HDAC1rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 5 pp 1241ndash1246 2004

[42] R Ju and M T Muller ldquoHistone deacetylase inhibitors activatep21WAF1 expression via ATMrdquo Cancer Research vol 63 no 11pp 2891ndash2897 2003

[43] P A Link M R Baer S R James D A Jones and A R KarpfldquoP53-inducible ribonucleotide reductase (P53R2RRM2B) isa DNA hypomethylation-independent decitabine gene targetthat correlates with clinical response in myelodysplastic syn-dromeacute myelogenous leukemiardquo Cancer Research vol 68no 22 pp 9358ndash9366 2008

[44] I Masgras S Carrera P J De Verdier et al ldquoReactive oxy-gen species and mitochondrial sensitivity to oxidative stressdetermine induction of cancer cell death by p21rdquo The Journalof Biological Chemistry vol 287 no 13 pp 9845ndash9854 2012

[45] J F Passos G Nelson C Wang et al ldquoFeedback between p21and reactive oxygen production is necessary for cell senes-cencerdquoMolecular Systems Biology vol 6 article 347 2010

[46] S Vijayaraghavalu J K Dermawan V Cheriyath and VLabhasetwar ldquoHighly synergistic effect of sequential treat-ment with epigenetic and anticancer drugs to overcome drugresistance in breast cancer cells is mediated via activation ofp21 gene expression leading to G2M cycle arrestrdquo MolecularPharmacology vol 10 pp 337ndash352 2013

[47] T Lu andT Finkel ldquoFree radicals and senescencerdquoExperimentalCell Research vol 314 no 9 pp 1918ndash1922 2008

[48] G H Stein L F Drullinger A Soulard and V Dulic ldquoDiffer-ential roles for cyclin-dependent kinase inhibitors p21 and p16in the mechanisms of senescence and differentiation in humanfibroblastsrdquo Molecular and Cellular Biology vol 19 no 3 pp2109ndash2117 1999

[49] Z Mao Z Ke V Gorbunova and A Seluanov ldquoReplicativelysenescent cells are arrested in G1 and G2 phasesrdquo Aging vol 4pp 431ndash435 2012

[50] S R Schwarze V X Fu J A Desotelle M L Kenowski andD F Jarrard ldquoThe identification of senescence-specific genesduring the induction of senescence in prostate cancer cellsrdquoNeoplasia vol 7 no 9 pp 816ndash823 2005

[51] S Amatori I Bagaloni D Viti and M Fanelli ldquoPrematuresenescence induced by DNA demethylating agent (Decitabine)as therapeutic option for malignant pleural mesotheliomardquoLung Cancer vol 71 no 1 pp 113ndash115 2011

[52] W Xu G Perez L Ngo C Gui and P A Marks ldquoInductionof polyploidy by histone deacetylase inhibitor a pathway forantitumor effectsrdquo Cancer Research vol 65 no 17 pp 7832ndash7839 2005

[53] T T Renault O Teijido B Antonsson L M Dejean and SManon ldquoRegulation of Bax mitochondrial localization by Bcl-2and Bcl-x(L) keep your friends close but your enemies closerrdquoThe International Journal of Biochemistry amp Cell Biology vol 45pp 64ndash67 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Acetylation equilibrium is maintained by balanced ratiobetween histone acetylases (HAT) and histone deacetylases(HDACs) action The activity of histones and many nonhi-stone proteins is regulated by the extent of acetylation oftheir lysine residues Dysfunction of acetylation process isoften associated with several diseases especially cancer andhistone deacetylase inhibitors (HDACi) are used to epige-netically correct aberrant HDAC activity [16 17] Changes ingene transcription direct induction of apoptosis productionof reactive oxygen species and induction of cell cycle arresthave been proposed as the mechanisms of HDACi action[18] Cell cycle arrest in G1 phase is widely documented asa consequence of HDACi-induced acetylation and transcrip-tion activation of p21WAF1 [19ndash21] The acetylation status ofp53 is extensively studied in connection with proapoptoticfunction of HDACi loss of acetylation completely abolishedp53-dependent growth arrest and apoptosis in HCT116 cells[22 23]

Methylation and acetylation mechanisms are often inter-connected and they occur ubiquitously depending on oneanother DNMT1 interacts with methyl-CpG binding pro-teins like MeCP2 which specifically recognizes fully methy-lated CpG sites or with MBD3 both forming complexeswith histone deacetylases HDAC1 and HDAC2 which inturn interact with DNMT1 [24 25] DNMT1 inhibitionwas also tested experimentally in cancer cells by alternatepathways using HDAC inhibitors that indirectly promoteubiquitin-dependent proteasomal degradation of DNMT1[26] Another mode of action of demethylating agents rep-resents the release of HDACs from gene promoters resultingin transcription activation This mechanism is responsiblefor cyclin-dependent kinase inhibitor p21WAF1 induction inAML-derived cells [27]Therefore combined action of drugsregulating methylation and acetylation is worth intensiveinvestigation

Combined treatment using two or more drugs is believedto bringmore targeted effect in cancer therapy Assessment ofconcurrent action of DNA methyltransferase inhibitor DACand histone deacetylase inhibitor suberoylanilide hydrox-amic acid (SAHA Vorinostat) showed cooperation betweenthese two drugs in suppressing colon carcinoma metasta-sis [28] or synergistic inhibition of Hey and SKOv3 cellgrowth by apoptosis and cell cycle arrest [25] Howeverlittle is known about apoptotic proteins expression dur-ing combined treatment Effect of such treatment on cellsoriginating from nonsolid tumors has not been tested yetalthough several clinical trials estimating combined effect ofDAC and SAHA administered concurrently or sequentiallyon different forms of MDS AML or ALL are currentlyin progress (httpwwwclinicaltrialsgov) In our previouswork we observed augmented effect of DAC and SAHA incombination on cell proliferation reactive oxygen speciesformation and apoptosis in leukemic cell line CML-T1 [2930] However drug concentrations used in this researchhave been shown to dramatically affect also the viabilityof peripheral blood lymphocytes (PBL) of healthy donorsTherefore action of low-drug concentrations inducing noor little adverse effect on normal cells has been studied inthis work The extent of apoptosis cell cycle distribution

p21WAF1 expression changes ROS formation and apoptoticproteins expression were investigated in leukemic cell lineswith different p53 status and in normal PBLThe mechanismof combined DAC and SAHA treatment with respect to thetumor suppressor p53 presence is discussed

2 Materials and Methods

21 Cell Culture and Chemicals Peripheral blood lympho-cytes of healthy donors were isolated from buffy coats onHistopaque 1077 (Sigma-Aldrich) as described earlier [31]Leukemia cell line CML-T1 and HL-60 (both from GermanCollection of Microorganisms and Cell Cultures Braun-schweig Germany) were cultivated in RPMI 1640 (BiochromAG Germany) supplemented with 10 FCS 37∘C and 5CO2atmosphere Decitabine (Sigma-Aldrich) and SAHA

(Cayman Chemical Company Ann Arbor MI USA) wereadded from 1mM stock solution to final concentration of1 120583M for different times up to 48 h Cytidine analogs azacyti-dine and decitabine are referred to be unstable in suspension[32] Therefore we investigated two modes of DAC actionin cell lines ldquoone-shotrdquo addition to final concentration of1 120583M for 48 h or sequential addition with fresh DAC addedafter 24 h Moreover two DAC concentrations were used in24 h sequential mode 05120583M to reach the same total drugexposure as with one-shot added 1120583M DAC or 1120583M tomaintain 1 120583M DAC concentration assuming that unstabledecitabine degraded in the first 24 h N-Acetyl-L-cysteine(NAC Sigma-Aldrich) and caspase inhibitors z-VAD-fmk(Santa Cruz) and Q-VD-OPh (RampD Systems) were added tofinal concentration of 10mM (NAC) 20120583M (z-VAD-fmk)and 10 120583M (Q-VD-OPh) respectively

22 Viability Test The viability (metabolic activity) of cellspreincubated in microplates with DAC andor SAHA for24 and 48 h was monitored using an MTT Kit I (RocheDiagnostics Corporation Indianapolis IN USA) Follow-ing the procedure described earlier [31] the absorbance ofreporter substrate was measured on an ELISA reader (MTXLab Systems Inc Vienna VA USA)

23 Flow Cytometry Distribution of cell cycle phases (mon-itored by propidium iodide PI) generation of reactive oxy-gen species (ROS observed by dichlorodihydrofluoresceindiacetate H

2DCFDA) mitochondrial membrane potential

(MMP measured by MitoTracker Red MTR) condensa-tion of chromatin (DNA stained by Hoechst 33342) andthe extent of apoptosis (defined by Annexin V-FITC posi-tivePI negative population of treated cells) were investigatedby flow cytometry All fluorescent probes were purchasedfrom Life Technologies Corporation All samples (50 000eventssample)were analyzed on anLSRFortessa cell analyzer(BD Biosciences)

231 Cell Cycle Cells treated by DAC andor SAHA for 24or 48 h were harvested washed twice with 2mL of PBSresuspended in 500120583L of PBS added into 45mL of ice-cold70 EtOH and stored in minus20∘C For the analysis suspension








2)







3 Results





0 10 20 30 40 50 600

20

40

60

80

100

120

Time (h)

Viab

ility

()

CML-T1

(a)

0 10 20 30 40 50 600

20

40

60

80

100

120

Time (h)

Viab

ility

()

HL-60

(b)

0 10 20 30 40 50 600

20

40

60

80

100

120

Time (h)

Viab

ility

()

PBL

(c)






CML-T1 HL-600

Frac

tion

of ce

lls (

)

5

10

15

20

25 lowast

lowastlowastlowast

lowastlowastlowast



(a)

CML-T1 HL-60

Frac

tion

of ce

lls (

)

0102030405060

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

CML-T1 HL-60

Frac

tion

of ce

lls (

)

0

50

100 lowastlowast


lowastlowastlowast

lowastlowastlowast

(c)

Caspase-3

Caspase-7

Procaspase

Procaspase

Active caspase

Active caspase

CML-T1 HL-60

SAHA

DACminus

minus minus


minusminus

+

++++

(d)

PARP

PBL PARP f116

f116

f88

f88

SAHADAC

CML-T1 HL-60

SAHADACminus

minus minus


minusminus

+

++++

minus

minus minus

+ minus +

++

(e)


Caspase-3

Caspase-7

Procaspase

Procaspase

Active caspase

Active caspase

SAHADAC

Q-VD-OPhz-VAD-fmk

minus

minus minus



minus+ + + +

+ +

+

+

+ +






2DCFDA fluorescence






SubG1 G2G1

0

20

40

60

80

100

C D S

S

DS C D S DS

Cel

l cyc

le p

hase

frac

tion

()

CML-T1 HL-60

(a)

p21

PBL p21

SAHADAC

CML-T1 HL-60

SAHA

DACminus

minus

+

+

minus

minus

+

minus + minus +

+

minus +minus +

minus +minus +

minus + minus +

120573-Actin

120573-Actin

(b)


CML-T1 HL-600

1

2

3

4

Mea

n flu

ores

cenc

e int

ensit

y (a

u)

lowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowast










CML-T1 HL-60

0

5

10

15

20

25

30

35

40

45Fr

actio

n of

cells

()

Frac

tion

of ce

lls (

)

0

10

20

30

40

50

60

lowast

lowast

C D

DD

1

DD

2

SAH

A

SAH

A+

DSA

HA+

DD

1SA

HA+

DD

2 C D

DD

1

DD

2

SAH

A

SAH

A+

DSA

HA+

DD

1SA

HA+

DD

2

(a)

CML-T1 HL-60

Procaspase

Procaspase

SAHA

Caspase-7

Caspase-3

1120583M DAC05120583M DAC



32kDa intermediate

17kDa fragment

minus


minus

minus

minus minus


minus

minus

+ minus +

+ +

+

+

+

minus + minus

minus + minus

+

+

minus minus + +

minus

minus

minus+

minus


minus

minus

minus minus


minus

minus

+ minus +

+ +

+

+

+

minus + minus

minus + minus

+

+

minus minus + +

minus

minus

minus+

(b)


CML-T1


1

2

3

5

15

Relat

ive e

xpre

ssio

n (a

u)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

(a)

HL-60


1

2

3

5

10

Relat

ive e

xpre

ssio

n (a

u) lowastlowast

lowastlowast

lowastlowastlowast


lowastlowastlowast

lowast

lowast

lowast

lowastlowast

(b)



p53

Puma

CML-T1 HL-60

SAHADAC

Bcl2

Mcl-1

BAX


minusminus

+

++minusminus ++

120573-Actin



4 Discussion






Anti-BAX

Intact

Anti-COX Merge

DAC +SAHA







5 Conclusion






Acknowledgments


References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








2)







3 Results





0 10 20 30 40 50 600

20

40

60

80

100

120

Time (h)

Viab

ility

()

CML-T1

(a)

0 10 20 30 40 50 600

20

40

60

80

100

120

Time (h)

Viab

ility

()

HL-60

(b)

0 10 20 30 40 50 600

20

40

60

80

100

120

Time (h)

Viab

ility

()

PBL

(c)






CML-T1 HL-600

Frac

tion

of ce

lls (

)

5

10

15

20

25 lowast

lowastlowastlowast

lowastlowastlowast



(a)

CML-T1 HL-60

Frac

tion

of ce

lls (

)

0102030405060

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

CML-T1 HL-60

Frac

tion

of ce

lls (

)

0

50

100 lowastlowast


lowastlowastlowast

lowastlowastlowast

(c)

Caspase-3

Caspase-7

Procaspase

Procaspase

Active caspase

Active caspase

CML-T1 HL-60

SAHA

DACminus

minus minus


minusminus

+

++++

(d)

PARP

PBL PARP f116

f116

f88

f88

SAHADAC

CML-T1 HL-60

SAHADACminus

minus minus


minusminus

+

++++

minus

minus minus

+ minus +

++

(e)


Caspase-3

Caspase-7

Procaspase

Procaspase

Active caspase

Active caspase

SAHADAC

Q-VD-OPhz-VAD-fmk

minus

minus minus



minus+ + + +

+ +

+

+

+ +






2DCFDA fluorescence






SubG1 G2G1

0

20

40

60

80

100

C D S

S

DS C D S DS

Cel

l cyc

le p

hase

frac

tion

()

CML-T1 HL-60

(a)

p21

PBL p21

SAHADAC

CML-T1 HL-60

SAHA

DACminus

minus

+

+

minus

minus

+

minus + minus +

+

minus +minus +

minus +minus +

minus + minus +

120573-Actin

120573-Actin

(b)


CML-T1 HL-600

1

2

3

4

Mea

n flu

ores

cenc

e int

ensit

y (a

u)

lowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowast










CML-T1 HL-60

0

5

10

15

20

25

30

35

40

45Fr

actio

n of

cells

()

Frac

tion

of ce

lls (

)

0

10

20

30

40

50

60

lowast

lowast

C D

DD

1

DD

2

SAH

A

SAH

A+

DSA

HA+

DD

1SA

HA+

DD

2 C D

DD

1

DD

2

SAH

A

SAH

A+

DSA

HA+

DD

1SA

HA+

DD

2

(a)

CML-T1 HL-60

Procaspase

Procaspase

SAHA

Caspase-7

Caspase-3

1120583M DAC05120583M DAC



32kDa intermediate

17kDa fragment

minus


minus

minus

minus minus


minus

minus

+ minus +

+ +

+

+

+

minus + minus

minus + minus

+

+

minus minus + +

minus

minus

minus+

minus


minus

minus

minus minus


minus

minus

+ minus +

+ +

+

+

+

minus + minus

minus + minus

+

+

minus minus + +

minus

minus

minus+

(b)


CML-T1


1

2

3

5

15

Relat

ive e

xpre

ssio

n (a

u)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

(a)

HL-60


1

2

3

5

10

Relat

ive e

xpre

ssio

n (a

u) lowastlowast

lowastlowast

lowastlowastlowast


lowastlowastlowast

lowast

lowast

lowast

lowastlowast

(b)



p53

Puma

CML-T1 HL-60

SAHADAC

Bcl2

Mcl-1

BAX


minusminus

+

++minusminus ++

120573-Actin



4 Discussion






Anti-BAX

Intact

Anti-COX Merge

DAC +SAHA







5 Conclusion






Acknowledgments


References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 10 20 30 40 50 600

20

40

60

80

100

120

Time (h)

Viab

ility

()

CML-T1

(a)

0 10 20 30 40 50 600

20

40

60

80

100

120

Time (h)

Viab

ility

()

HL-60

(b)

0 10 20 30 40 50 600

20

40

60

80

100

120

Time (h)

Viab

ility

()

PBL

(c)






CML-T1 HL-600

Frac

tion

of ce

lls (

)

5

10

15

20

25 lowast

lowastlowastlowast

lowastlowastlowast



(a)

CML-T1 HL-60

Frac

tion

of ce

lls (

)

0102030405060

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

CML-T1 HL-60

Frac

tion

of ce

lls (

)

0

50

100 lowastlowast


lowastlowastlowast

lowastlowastlowast

(c)

Caspase-3

Caspase-7

Procaspase

Procaspase

Active caspase

Active caspase

CML-T1 HL-60

SAHA

DACminus

minus minus


minusminus

+

++++

(d)

PARP

PBL PARP f116

f116

f88

f88

SAHADAC

CML-T1 HL-60

SAHADACminus

minus minus


minusminus

+

++++

minus

minus minus

+ minus +

++

(e)


Caspase-3

Caspase-7

Procaspase

Procaspase

Active caspase

Active caspase

SAHADAC

Q-VD-OPhz-VAD-fmk

minus

minus minus



minus+ + + +

+ +

+

+

+ +






2DCFDA fluorescence






SubG1 G2G1

0

20

40

60

80

100

C D S

S

DS C D S DS

Cel

l cyc

le p

hase

frac

tion

()

CML-T1 HL-60

(a)

p21

PBL p21

SAHADAC

CML-T1 HL-60

SAHA

DACminus

minus

+

+

minus

minus

+

minus + minus +

+

minus +minus +

minus +minus +

minus + minus +

120573-Actin

120573-Actin

(b)


CML-T1 HL-600

1

2

3

4

Mea

n flu

ores

cenc

e int

ensit

y (a

u)

lowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowast










CML-T1 HL-60

0

5

10

15

20

25

30

35

40

45Fr

actio

n of

cells

()

Frac

tion

of ce

lls (

)

0

10

20

30

40

50

60

lowast

lowast

C D

DD

1

DD

2

SAH

A

SAH

A+

DSA

HA+

DD

1SA

HA+

DD

2 C D

DD

1

DD

2

SAH

A

SAH

A+

DSA

HA+

DD

1SA

HA+

DD

2

(a)

CML-T1 HL-60

Procaspase

Procaspase

SAHA

Caspase-7

Caspase-3

1120583M DAC05120583M DAC



32kDa intermediate

17kDa fragment

minus


minus

minus

minus minus


minus

minus

+ minus +

+ +

+

+

+

minus + minus

minus + minus

+

+

minus minus + +

minus

minus

minus+

minus


minus

minus

minus minus


minus

minus

+ minus +

+ +

+

+

+

minus + minus

minus + minus

+

+

minus minus + +

minus

minus

minus+

(b)


CML-T1


1

2

3

5

15

Relat

ive e

xpre

ssio

n (a

u)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

(a)

HL-60


1

2

3

5

10

Relat

ive e

xpre

ssio

n (a

u) lowastlowast

lowastlowast

lowastlowastlowast


lowastlowastlowast

lowast

lowast

lowast

lowastlowast

(b)



p53

Puma

CML-T1 HL-60

SAHADAC

Bcl2

Mcl-1

BAX


minusminus

+

++minusminus ++

120573-Actin



4 Discussion






Anti-BAX

Intact

Anti-COX Merge

DAC +SAHA







5 Conclusion






Acknowledgments


References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


CML-T1 HL-600

Frac

tion

of ce

lls (

)

5

10

15

20

25 lowast

lowastlowastlowast

lowastlowastlowast



(a)

CML-T1 HL-60

Frac

tion

of ce

lls (

)

0102030405060

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

CML-T1 HL-60

Frac

tion

of ce

lls (

)

0

50

100 lowastlowast


lowastlowastlowast

lowastlowastlowast

(c)

Caspase-3

Caspase-7

Procaspase

Procaspase

Active caspase

Active caspase

CML-T1 HL-60

SAHA

DACminus

minus minus


minusminus

+

++++

(d)

PARP

PBL PARP f116

f116

f88

f88

SAHADAC

CML-T1 HL-60

SAHADACminus

minus minus


minusminus

+

++++

minus

minus minus

+ minus +

++

(e)


Caspase-3

Caspase-7

Procaspase

Procaspase

Active caspase

Active caspase

SAHADAC

Q-VD-OPhz-VAD-fmk

minus

minus minus



minus+ + + +

+ +

+

+

+ +






2DCFDA fluorescence






SubG1 G2G1

0

20

40

60

80

100

C D S

S

DS C D S DS

Cel

l cyc

le p

hase

frac

tion

()

CML-T1 HL-60

(a)

p21

PBL p21

SAHADAC

CML-T1 HL-60

SAHA

DACminus

minus

+

+

minus

minus

+

minus + minus +

+

minus +minus +

minus +minus +

minus + minus +

120573-Actin

120573-Actin

(b)


CML-T1 HL-600

1

2

3

4

Mea

n flu

ores

cenc

e int

ensit

y (a

u)

lowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowast










CML-T1 HL-60

0

5

10

15

20

25

30

35

40

45Fr

actio

n of

cells

()

Frac

tion

of ce

lls (

)

0

10

20

30

40

50

60

lowast

lowast

C D

DD

1

DD

2

SAH

A

SAH

A+

DSA

HA+

DD

1SA

HA+

DD

2 C D

DD

1

DD

2

SAH

A

SAH

A+

DSA

HA+

DD

1SA

HA+

DD

2

(a)

CML-T1 HL-60

Procaspase

Procaspase

SAHA

Caspase-7

Caspase-3

1120583M DAC05120583M DAC



32kDa intermediate

17kDa fragment

minus


minus

minus

minus minus


minus

minus

+ minus +

+ +

+

+

+

minus + minus

minus + minus

+

+

minus minus + +

minus

minus

minus+

minus


minus

minus

minus minus


minus

minus

+ minus +

+ +

+

+

+

minus + minus

minus + minus

+

+

minus minus + +

minus

minus

minus+

(b)


CML-T1


1

2

3

5

15

Relat

ive e

xpre

ssio

n (a

u)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

(a)

HL-60


1

2

3

5

10

Relat

ive e

xpre

ssio

n (a

u) lowastlowast

lowastlowast

lowastlowastlowast


lowastlowastlowast

lowast

lowast

lowast

lowastlowast

(b)



p53

Puma

CML-T1 HL-60

SAHADAC

Bcl2

Mcl-1

BAX


minusminus

+

++minusminus ++

120573-Actin



4 Discussion






Anti-BAX

Intact

Anti-COX Merge

DAC +SAHA







5 Conclusion






Acknowledgments


References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


SubG1 G2G1

0

20

40

60

80

100

C D S

S

DS C D S DS

Cel

l cyc

le p

hase

frac

tion

()

CML-T1 HL-60

(a)

p21

PBL p21

SAHADAC

CML-T1 HL-60

SAHA

DACminus

minus

+

+

minus

minus

+

minus + minus +

+

minus +minus +

minus +minus +

minus + minus +

120573-Actin

120573-Actin

(b)


CML-T1 HL-600

1

2

3

4

Mea

n flu

ores

cenc

e int

ensit

y (a

u)

lowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowast










CML-T1 HL-60

0

5

10

15

20

25

30

35

40

45Fr

actio

n of

cells

()

Frac

tion

of ce

lls (

)

0

10

20

30

40

50

60

lowast

lowast

C D

DD

1

DD

2

SAH

A

SAH

A+

DSA

HA+

DD

1SA

HA+

DD

2 C D

DD

1

DD

2

SAH

A

SAH

A+

DSA

HA+

DD

1SA

HA+

DD

2

(a)

CML-T1 HL-60

Procaspase

Procaspase

SAHA

Caspase-7

Caspase-3

1120583M DAC05120583M DAC



32kDa intermediate

17kDa fragment

minus


minus

minus

minus minus


minus

minus

+ minus +

+ +

+

+

+

minus + minus

minus + minus

+

+

minus minus + +

minus

minus

minus+

minus


minus

minus

minus minus


minus

minus

+ minus +

+ +

+

+

+

minus + minus

minus + minus

+

+

minus minus + +

minus

minus

minus+

(b)


CML-T1


1

2

3

5

15

Relat

ive e

xpre

ssio

n (a

u)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

(a)

HL-60


1

2

3

5

10

Relat

ive e

xpre

ssio

n (a

u) lowastlowast

lowastlowast

lowastlowastlowast


lowastlowastlowast

lowast

lowast

lowast

lowastlowast

(b)



p53

Puma

CML-T1 HL-60

SAHADAC

Bcl2

Mcl-1

BAX


minusminus

+

++minusminus ++

120573-Actin



4 Discussion






Anti-BAX

Intact

Anti-COX Merge

DAC +SAHA







5 Conclusion






Acknowledgments


References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


CML-T1 HL-60

0

5

10

15

20

25

30

35

40

45Fr

actio

n of

cells

()

Frac

tion

of ce

lls (

)

0

10

20

30

40

50

60

lowast

lowast

C D

DD

1

DD

2

SAH

A

SAH

A+

DSA

HA+

DD

1SA

HA+

DD

2 C D

DD

1

DD

2

SAH

A

SAH

A+

DSA

HA+

DD

1SA

HA+

DD

2

(a)

CML-T1 HL-60

Procaspase

Procaspase

SAHA

Caspase-7

Caspase-3

1120583M DAC05120583M DAC



32kDa intermediate

17kDa fragment

minus


minus

minus

minus minus


minus

minus

+ minus +

+ +

+

+

+

minus + minus

minus + minus

+

+

minus minus + +

minus

minus

minus+

minus


minus

minus

minus minus


minus

minus

+ minus +

+ +

+

+

+

minus + minus

minus + minus

+

+

minus minus + +

minus

minus

minus+

(b)


CML-T1


1

2

3

5

15

Relat

ive e

xpre

ssio

n (a

u)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

(a)

HL-60


1

2

3

5

10

Relat

ive e

xpre

ssio

n (a

u) lowastlowast

lowastlowast

lowastlowastlowast


lowastlowastlowast

lowast

lowast

lowast

lowastlowast

(b)



p53

Puma

CML-T1 HL-60

SAHADAC

Bcl2

Mcl-1

BAX


minusminus

+

++minusminus ++

120573-Actin



4 Discussion






Anti-BAX

Intact

Anti-COX Merge

DAC +SAHA







5 Conclusion






Acknowledgments


References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


p53

Puma

CML-T1 HL-60

SAHADAC

Bcl2

Mcl-1

BAX


minusminus

+

++minusminus ++

120573-Actin



4 Discussion






Anti-BAX

Intact

Anti-COX Merge

DAC +SAHA







5 Conclusion






Acknowledgments


References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Anti-BAX

Intact

Anti-COX Merge

DAC +SAHA







5 Conclusion






Acknowledgments


References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgments


References































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

combined treatment with low concentrations of decitabine and

Documents